Uses of chemically-modified cholinesterases for detoxification of organophosporus compounds

ABSTRACT

A circulatory long-lived cholinesterase (ChE) protein, such as acetylcholinesterase (AChE) or butyrylcholinesterase (BChE), which is a ChE protein modified with a non-antigenic polymer. The ChE may be AChE, such as native AChE of mammalian origin or of non-mammalian origin, or recombinant AChE. The recombinant AChE may be mutated at one or more amino-acid residues. The BChE may be native BChE of mammalian origin or of non-mammalian origin.

FIELD OF THE INVENTION

The present invention relates to the chemical modification ofcholinesterases (ChEs) by polyethylene glycol (PEG), to proteins ofimproved stability and circulatory half-life obtained thereby, topharmaceutical comprising them, and their uses.

BACKGROUND OF THE INVENTION

Conjugating biologically active proteins to polymers has been shown toimprove the circulating life of the administered protein and to reduceits antigenicity and immunogenicity. For example, U.S. Pat. No.4,179,337 discloses the use of PEG or polypropylene glycol coupled toproteins to provide a physiologically active non-immunogenic watersoluble polypeptide composition. Conjugates are formed by reacting abiologically active material with a several fold molar excess of apolymer which has been modified to contain a terminal linking group.

A variety of means have been used to attach polyethylene glycolmolecules to the protein. For example, U.S. Pat. No. 5,932,464 and U.S.Pat. No. 5,990,237 disclose methods for coupling polyethylene glycol toa biomaterial. Generally, polyethylene glycol molecules are connected toa protein via a reactive group found thereon. Amino groups, such asthose on lysine residues or at the N-terminus, as well as thiol groupson cysteine, or other reactive groups on protein surface, are convenientfor such attachment. For many biologically active materials, however,the conjugation process is accompanied by several complications.Firstly, it is not always specific with regard to attachment sites.Secondly, loss of biological activity is often caused by the conjugationreaction. For example, if too much of the activated polymer is attachedto the target protein or polypeptide, biological activity can beseverely reduced or lost. Furthermore, if the wrong linker joining thepolymer to the protein is used, or if an insufficient amount of polymeris attached to the target, the therapeutic value of the resultantconjugate is limited. Often, such conjugates do not demonstrate enoughof an increase in the circulating life to compensate for the loss inbioactivity. Problems can also result when a therapeutic moiety's activesite (i.e. where groups associated with bioactivity are located) becomessterically blocked as a result of the polymer attachment. Accordingly,the outcome of a protein conjugation process is unpredictable in nature.

Cholinesterases are important proteins. Acetylcholinesterase (AChE, EC3.1.1.7) plays a pivotal role in the cholinergic system where itfunctions in the rapid termination of nerve impulse transmission. Thefunction of the related enzyme butyrylcholinesterase (BChE, EC 3.1.1.8)is yet unknown, nor is its specific natural substrate known, but it iscapable of hydrolysing acetylcholine. It has been suggested that BChEacts as an endogenous scavenging enzyme important for the detoxificationof natural poisons [Massoulie, J., et al., (1993) Prog. Brain Res. 98,139-146]. The high reactivity of these enzymes toward organophosphorus(OP) compounds, makes exogenous cholinesterase an effective therapeuticagent in the prophylaxis and treatment of OP-poisoning. Indeed thesuccessful exploitation of the scavenging potential of various forms ofcholinesterases which include fetal bovine AChE [Maxwell, D. M., et al.,(1992) Toxicol. Appl. Pharmacol. 115, 44-49], human BChE [Raveh, L., etal., (1993) Biochem. Pharmacol. 45, 37-41], equine BChE [Broomfield, C.A., et al., (1991) J. Pharmacol. Exp. Ther. 259, 683-698] has beendemonstrated in rodents [Raveh, L., et al., ibid] and in non-humanprimates [Broomfield, C A, et al., ibid; Maxwell D M et al., ibid] andeven for treatment of humans exposed to organophosphate pesticides[Cascio, C., et al., (1988) Minerva Anestesiol. 54, 337-338].

The use of ChE as a biological scavenger requires sources for largequantities of purified enzyme and depends on the retention of the enzymein the circulation for sufficiently long periods of time. Production ofAChE in various expression systems is known. However, the successfulapplication of recombinant ChE as a bioscavenging agent of therapeuticvalue requires its retention within the circulation for appreciableperiods of time. Examination of the pharmacokinetic profile of variousrecombinant AChEs demonstrates that these are eliminated from thecirculation rapidly, displaying mean residence time values (MRT) of5-100 minutes [Kronman, C., et al., (1992) Gene 121, 295-304; Chitlaru,T., et al., (1998) Biochem. J. 336, 647-658], and therefore do not meetthe requirements for OP bioscavenging in their non-modified state.

It would therefore be desirable to be able to provide ChEs that exhibitimproved retention within the circulation, which could be used astherapeutic bioscavenging agents. It has now been surprisingly found,and this is an object of the present invention, that it is possible toprovide such improved modified ChEs, by a modification made usingpolyethylene glycol groups attached to the lysine moieties located onthe ChEs.

It is thus an object of the invention to provide such modified ChEswhich exhibit excellent and unprecedented circulatory longevity.

It is another object of the invention to provide a beneficial extensionof circulatory residence by polyethylene glycol appendage, whichoverrides the various deleterious factors which contribute to the rapidclearance of recombinant AChEs, allowing long-term circulatory residenceof recombinant AChE molecules which are devoid of glycans, or containglycans devoid of sialic acid capping, or do not assemble intomultimeric forms.

It is a further object of the invention to providepharmaceutically-compatible AChEs from a wide variety of sources,including those which display suboptimal post-translation processing.

It is a further object of the invention to provide a novel use of themodified AChEs of the invention in the prophylactic or acute treatmentof OP poisoning.

Other objects and advantages of the invention will become apparent asthe description proceeds.

SUMMARY OF THE INVENTION

The present invention relates to a novel class of cholinesterasesderivatives wherein the cholinesterase molecules are attached to a watersoluble polymer, to a method for preparing such derivatives, and totheir uses.

In one aspect the invention is directed to a circulatory long-livedcholinesterases (ChEs) protein, which is a ChEs protein modified with anon-antigenic polymer. According to a preferred embodiment of theinvention, the ChE is acetylcholinesterase (AChE). According to anotherpreferred embodiment of the invention, the ChE is butyrylcholinesterase(BChE). As used herein, the term “circulatory long-lived protein” meansa protein which, when administered to a subject in vivo, has a meanresidence-time in the body of at least several hours, e.g., more thantwo hours. It should be noted that non-modified recombinant ChEsproteins of the prior art may exhibit very low circulatory meanresidence-times, of the order of minutes.

The ChE can be of various origins. For instance, it can be native ChE ofmammalian or non-mammalian origin, or recombinant ChE, and it can befurther mutated at one or more amino-acid residues.

The non-antigenic polymer is preferably selected from the groupconsisting of dextran, polyvinyl pyrrolidones, polyacrylamides,polyvinyl alcohols and carbohydrate-based polymers and it preferablycomprises a polyalkylene oxide. A preferred polyalkylene oxide is apolyethylene glycol (PEG), such as mono-methoxy-PEG. Furthermore the PEGcan be chemically-activated-PEG, such as succinimidyl derivative of PEGpropionic acid (SPA-PEG).

The PEG preferred for use in the invention is of molecular weight fromabout 200 to about 100,000 dalton, preferably from about 2000 to about40,000 dalton, and more preferably from about 5000 to about 20,000dalton.

The polymer can be conjugated in different ways, e.g., it can beconjugated through primary amines, carboxyl sites, thiol groups orcarbohydrates.

In another aspect, the invention is directed to the use of aphysiologically active, long-lived non-antigenic polymer conjugatedcholinesterase (ChE) proteins, as an organophosphates scavenger.

The invention further encompasses a pharmaceutical preparationcomprising physiologically active, long-lived non-antigenic polymerconjugated cholinesterases (ChEs) proteins, and its use as anorganophosphates scavenger.

The invention is also directed to a method for increasing thecirculatory half-life of a physiologically active cholinesterase (ChE)protein in vivo, comprising conjugating said ChE with a non-antigenicpolymer.

Though cholinesterases from natural sources or from recombinantproduction systems can potentially serve as bioscavengers of OPcompounds, and the invention is by no means limited to the use of ChEsfrom any specific source, recombinant versions of ChE represent apreferable mode for the formulation of a therapeutical bioscavenger dueto the fact that recombinant heterologous production systems enable theintroduction of bioscavenging-favorable modifications by site-directedmutagenesis of the ChE genes prior to their introduction into the hostcells. Thus, for example, mutations which altered the catalyticperformance and which result in the generation of an enzyme form whichis less susceptible to irreversible inactivation (aging) can beadvantageously used, such as those that were introduced into recombinantChE, enhancing the bioscavenging potential of the enzymes [Shafferman,A., et al. (1992), J. Biol. Chem. 267, 17640-17648; Shafferman, A., etal. (1993), Proceedings of Medical Defense Bioscience Review, Vol. 3,1111-1124; Shafferman, A., et al. (1996), Proceedings of Medical DefenseBioscience Review, Vol. 1, 23-32; Ordentlich, A., et al. (1996),Proceedings of Medical Defense Bioscience Review, Vol. 1, 231-239].

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the analysis of PEG-AChE products by SDS-PAGE;

FIG. 2 shows the pharmacokinetic profiles of non-modified andPEG-modified AChE;

FIG. 3 shows the correlation of MRT and the molecular weight of theconjugated proteins;

FIG. 4 shows the pharmacokinetic profiles of non-PEGylated and PEGylatedFBS-AChE;

FIG. 5 Shows the sucrose gradient sedimentation profiles of ΔC-rHuAChEand FBS-AChE;

FIG. 6 shows the glycan analysis of the desialylated, over-sialylatedand native recombinant human AChE;

FIG. 7 shows the pharmacokinetic profiles of non-PEGylated and PEGylateddesialylated-AChE, over-sialylated-AChE and native AChE;

FIG. 8 shows the pharmacokinetic profiles of non-PEGylated and PEGylateddeglycosylated-AChE;

FIG. 9 shows the glycan analysis of WT and N350Q/N464Q mutatedrecombinant human AChE;

FIG. 10 shows the pharmacokinetic profiles of non-PEGylated andPEGylated N350Q/N464Q-AchE; and

FIG. 11 shows the pharmacokinetic profiles of non-PEGylated andPEGylated BChE (partially sialylated).

DETAILED DESCRIPTION OF THE INVENTION General Methods and Procedures

1. Cholinesterases

As stated, the present invention is not limited to the use with anyparticular ChE, and ChEs prepared by various methods can be used. Themethods for the preparation of ChEs are well known in the art. Forexample, AChE or BChE may be prepared by a process comprising the stepsof: Extracting from an organ (such as liver, spleen, lung, bone marrow,brain, kidney, placenta and the like), blood cells (such as platelets,white blood cells and the like), plasma, serum and the like, of a mammal(such as rat, bovine, horse, sheep and the like) or non-mammal (such astorpedo, electric eel and the like), and purification thereof, astaught, for instance, in Velan, B., et al. (1991), J. Biol. Chem. 266,23977-23984, in Kronman, C., et al. (1992), Gene 121, 295-304, or inLazar, A., et al. (1993), Cytotechnology 13, 115-123.

AChE or BChE may also be prepared by genetic engineering methods, e.g.by inserting a gene encoding AChE or BChE into an appropriate vector,transfecting a host cell by inserting said inserted vector, andpurifying the enzyme from the cell extract or from the supernatant fluidof the cultured transfected cells, as discussed in the aforementionedVelan, Kronman and Lazar references. The host cell employed is notlimited to any specific cell, and various host cells conventionally usedin genetic engineering methods can be used, which are, for example,Escherichia coli, Bacillus subtilis, yeast, mold fungi, plant or animalcells and the like. A more specific process for the preparation of AChEor BChE from animal cells comprises the steps of: Transforming an animalcell (such as HEK-293 cells, Chinese Hamster Ovary (CHO) cells, mouseC127 cells, monkey COS cells, Sf (Spodoptera frugiperda) cells and thelike) with a gene encoding amino acid sequence of AChE or BChE; andpurifying the enzyme from the cell extract or from the supernatant fluidof the cultured cells.

AChE or BChE prepared by the above processes include any AChE or BChEthat has substantially the same activities such as a partial deletionderivative of the amino acid sequence, a substitution derivative of anamino acid, an insertion derivative of other amino acid sequences, aderivative from binding one or more amino acids to N- or C-terminus ofthe amino acid sequence, or sugar chain deletion or insertion orsubstitution derivatives.

2. Polyethylene Glycols

“Polyethylene glycol” or “PEG” refers to mixtures of condensationpolymers of ethylene oxide and water, in a branched or straight chain,represented by the general formula H(OCH₂ CH₂)_(n)OH, wherein n is atleast 4. “Polyethylene glycol” or “PEG” is used in combination with anumeric suffix to indicate the approximate average molecular weightthereof. For example, PEG-5,000 refers to polyethylene glycol having atotal average molecular weight of about 5,000; PEG-20,000 refers topolyethylene glycol having a total average molecular weight of about20,000.

To conjugate the ChE to polymers such as poly(alkylene oxides), one ofthe polymer hydroxyl end-groups is converted into a reactive functionalgroup which allows conjugation. This process is frequently referred toas “activation” and the product is called an “activated” polymer oractivated poly(alkylene oxide). Other substantially non-antigenicpolymers are similarly “activated” or functionalized. The activatedpolymers are reacted with AChE or BChE so that attachment occurs atε-amino groups of lysines, or at the N-terminal amino group. Freecarboxylic acid groups, suitably activated carbonyl groups, oxidizedcarbohydrate moieties and mercapto groups if available on the ChE canalso be used as supplemental or alternative attachment sites, ifdesired. Among the substantially non-antigenic polymers, mono-activated,alkoxy-terminated polyalkylene oxides (PAO's), such asmonomethoxy-terminated polyethylene glycols (mPEG's) are preferred.

Suitable polymers will vary substantially by weight. Polymers havingmolecular number average weights ranging from about 2000 to about 40,000are usually selected for the purposes of the present invention.Molecular weights of from about 5,000 to about 20,000 are particularlypreferred.

As an alternative to PAO-based polymers, effectively non-antigenicmaterials such as dextran, polyvinyl pyrrolidones, polyacrylamides suchas HPMA's-hydroxypropylmethacrylamides, polyvinyl alcohols,carbohydrate-based polymers, copolymers of the foregoing, and the likecan be used.

Those of ordinary skill in the art will realize that the foregoing listis merely illustrative and that all polymer materials having thequalities described herein are contemplated as polymers useful in theinvention. For purposes of the present invention, “substantially oreffectively non-antigenic” means all materials understood in the art asbeing nontoxic and not eliciting an appreciable immunogenic response inmammals.

General methods of attaching polyethylene glycol to proteins aredisclosed, e.g., in U.S. Pat. No. 4,179,337, the description of which isincorporated herein by reference. Other methods of attachingpolyethylene glycol are well known in the art, e.g., from U.S. Pat. No.5,122,614, which is also incorporated herein by reference. Therefore,these methods are not discussed herein in detail, for the sake ofbrevity.

3. Reaction Conditions

Conjugation reactions, sometimes referred to as PEGylation reactions,are often carried out in solution without regard to where the polymerwill attach to the protein. Such techniques are also usually carried outat slightly alkaline pH, i.e. pH 7 to about 9.

The processes of the present invention therefore includes reacting asolution containing AChE or BChE with a suitable amount of amono-functional methoxy-activated polymer such as succinimidylderivative of PEG propionic acid (SPA-PEG; Shearwater Polymers, Inc.) ata pH which is sufficient to facilitate covalent attachment of at least aportion of the polymer strands to primary amines, such as the ε-amine oflysine residues or to the N-terminus of the individual AChE or BChEmolecules. A preferred (but not limitative) pH is about 8.0.

Conjugation is typically carried out by conducting the attachmentreaction with a molar excess of the activated polymer with respect tothe primary amines in AChE or BChE. In this regard, the process istypically—but non-limitatively—carried out with about 5 to 400-foldmolar excesses, preferably about 20-200-fold molar excesses, and mostpreferably about 50-100-fold-molar excesses. The conjugation reactioncan be conveniently carried out at about room temperature. It is alsopreferred that the coupling reaction be allowed to proceed for rathershort periods of time, i.e. 1-2 hours. In practice, the reactionconditions yield a mixture of polymer-ChE positional isomers.Preferably, each isomer contains several polymer strands attached to theAChE or BChE via an amino acid residue. As will be understood by theskilled person, alternative ChEs (such as BChE) or different AChEs (suchas AChE from bovine or other sources or genetically modified version ofthe enzymes) will provide alternative distributions of positionalisomers, depending upon the amino acid sequence of the startingmaterial. Due to the nature of the solution-based conjugation reactions,the compositions are a heterogeneous mixture of species which containsthe polymer strand(s) attached at different sites on the ChE molecule.Given that there are multiple possible attachment points for a polymerto an AChE or BChE molecule and given the range of acceptable molarratios, it will be understood that, in certain embodiments, theconjugate product includes one or more polymeric strands. In suchembodiments, the substitutions may range from 1 to about 11 polymers perAChE molecule and from 1 to about 40 polymers per BChE molecule. As willbe appreciated by persons skilled in the art, the number of PEGconjugated to ChEs can be controlled in different ways, e.g., by the useof different ChE species, which vary in the number of their lysineresidue contents, by the use of mutants in which the number of lysineresidues was reduced by site directed mutagenesis, and by the use ofother attachment sites such as unique cysteines, as targets forconjugation.

EXAMPLES

All the above and other characteristics and advantages of the inventionwill be further understood through the following illustrative andnon-limitative examples.

Example 1 Preparation of Recombinant Human AChE

A C-terminal truncated version of recombinant human AChE was prepared,to be used for conjugation. Truncation of the C-terminus (a substitutionof the last 40 amino acids with a pentapeptide, ASEAP) of the T-subunitof human AChE [Soreq, H., et al. (1990), Proc. Natl. Acad. Sci. USA 87,9688-9692; accession number for human AChE M55040], was preformed by DNAcassette replacement [Shafferman, A., et al., (1992), J. Biol. Chem.267, 17640-17648], as described recently [Kryger, G., et al. (2000),Act. Cryst. D56, 1385-1394]. The DNA coding sequences for the truncatedHuAChE (ΔC-HuAChE) was inserted into a tripartite expression vectorexpressing also the reporter gene cat and the selection marker neo[Velan, B., et al. (1991), J. Biol. Chem. 266, 23977-23984; Kronman, C.,et al. (1992), Gene 121, 295-304]. Generation of stably transfectedHEK-293 cell lines expressing high levels of rHuAChE and purification ofthe secreted enzyme was performed as described previously [Velan B.,ibid; Kronman C., ibid].

Example 2

Attachment of PEG chains to primary amines in rHuAChE was performedusing succinimidyl propionate activated methoxy PEG (SPA-PEG; Shearwaterpolymers, Inc.). Purified ΔC-HuAChE resulting from example 1 (1-5 μM)was incubated with PEG-5000 or PEG-20000 in 50 mM phosphate buffer pH8.0 for 2 hours at room temperature. PEG was added at a ratio of 5:1(lowratio) or 25:1 (high ratio) [PEG]₀/[AChE primary amines]₀. The modifiedproducts were dialyzed extensively against phosphate buffer saline(PBS). Samples of the proteins were resolved on 7.5% SDS-polyacrylamidegels, electrotransfered onto nitrocellulose and subjected toWestern-blot analysis using mouse polyclonal anti-HuAChE antibodies[Shafferman, A. et al., ibid].

The results of the SDS-PAGE analysis are set forth in FIG. 1 which showsthe unique migration pattern of discrete bands of the PEG-AChE productsgenerated under the various conditions. It is clear from these resultsthat increasing the PEG to AChE ratio leads to a higher level of lysineoccupancy by PEG, and that the use of PEG of higher molecular weightleads to generation of higher molecular weight conjugation products. Itshould be noted that delicate tuning of conjugation conditions resultedin relatively homogenous products (no more than 2-3 differentlyPEGylated forms in each preparation).

Example 3

Measurement of the kinetic parameters, as well as inhibition constantsof non-modified and PEG-modified AChE demonstrated that, enzymaticperformance of AChE was not affected by PEG-conjugation. This issurprising since, for many proteins, PEG conjugation leads to areduction or loss of their biological activity [Monfardini, C. andVeronese, F. M. (1998), Bioconjug. Chem. 9, 418-450; Francis, G. E., etal. (1998), Inter. J. Hemato. 68, 1-18].

AChE activity was measured according to Ellman et al. [Ellman, G. L., etal. (1961), Biochem. Pharmacol. 7, 88-95]. Assays were performed in thepresence of 0.5 mM acetylthiocholine, 50 mM sodium phosphate buffer pH8.0, 0.1 mg/ml BSA and 0.3 mM 5,5′-dithiobis-(2-nitrobenzoic acid). Theassay was carried out at 27° C. and monitored by a Thermomax microplatereader (Molecular Devices). K_(m) values of HuAChE and PEG-HuAChE foracetylthiocholine were obtained from Lineweaver-Burk plots and k_(cat)calculations were based on active-site titration [Shafferman, A. et al.,ibid]. Interactions of HuAChE or PEG-HuAChE with the AChE-specificinhibitors edrophonium, propidium, BW284C51, snake-venomtoxin—fasciculin-II and with the organophosphate compounddiisopropylfluorophosphate (DFP) were analyzed as described previously[Ordentlich, A., et al. (1996), Proceedings of Medical DefenseBioscience Review, Vol. 1, 231-239]. The comparative results are setforth in the following tables, where Table 1 shows a comparison ofcatalytic properties of non-modified and PEG-modified ΔC-AChE, and Table2 is a comparison of inhibition constants of non-modified andPEG-modified C-AChE towards various inhibitors.

TABLE 1 AChE Preparations ΔC-AChE-PEG- Kinetic ΔC-AChE-PEG- 20000parameters ΔC-AChE 20000 low ratio high ratio K_(m) 0.09 ± 0.01 0.09 ±0.01 0.09 ± 0.01 (mM) k_(cat) 3.9 ± 0.2 4.0 ± 0.2 3.9 ± .01 (10⁵ ×min⁻¹) K_(app) 43 ± 2  44 ± 4  43 ± 3  (10⁸ × M⁻¹min⁻¹) K_(ss) 9 ± 2 6 ±2 10 ± 3  (mM) *Values are means ± S.D. for at least three independentexperiments

TABLE 2 AChE Preparations ΔC-AChE-PEG- ΔC-AChE-PEG- 20000 InhibitorΔC-AChE 20000 low ratio high ratio Edrophonium 0.8 ± 0.2 0.8 ± 0.2 0.8 ±0.2 K_(i) (μM) Propidium 1.0 ± 0.3 0.6 ± 0.2 0.7 ± 0.3 K_(i) (μM)BW284C51 8 ± 1 10 ± 2  6 ± 1 K_(i) (nM) Fasciculin 0.8 ± 0.1 0.7 ± 0.21.1 ± 0.3 K_(i) (nM) DFP 57 ± 4  58 ± 3  51 ± 2  k_(i) (10⁻⁴ × M⁻¹min⁻¹)*Values of inhibition constants are means ± S.D. for at least threeindependent experiments

From the foregoing, it can be seen that the K_(m) and the k_(cat) valuesof the modified enzymes were indistinguishable, within the experimentalerror, from those of the non-modified enzyme (Table 1). Likewise, theinhibition constants (K_(i)) for the classical non-covalent active-siteinhibitors or the covalent organophosphate DFP were similar to those ofthe non-modified AChE (Table 2). Thus it is seen that PEGylation ofΔC-HuAChE can be optimized, to be compatible with maintenance of fullenzymatic activity with no apparent effect on reactivity toward variousligands or on the scavenging potential of toxic agents exemplified bythe organophosphate diisopropylfluorophosphate (DFP).

Example 4

The pharmacokinetic profiles of non-modified and various preparations ofPEG-modified AChE were resolved. Clearance experiments in mice (3 to 6ICR male mice per enzyme sample) and analysis of pharmacokineticprofiles were carried out as described essentially previously [Kronman,C., et al. (1992), Gene 121, 295-304]. The study was approved by thelocal ethical committee on animal experiments. Mice were injected withthe various rHuAChE preparations (40 μg/mouse in 0.2 ml PBS). ResidualAChE activity in blood samples was measured and all values werecorrected for background hydrolytic activity in the blood (using sampleswithdrawn 1 hour before performing the experiment). AChE activity valuesin samples removed immediately after injection were assigned a value of100% and used for calculation of residual activity. Backgroundcholinesterase levels in blood of pre-administered mice were less than 2units/ml. The clearance patterns of the various enzyme preparations wereusually biphasic and fitted to a bi-exponential eliminationpharmacokinetic model (C_(t)=Ae^(−k) ^(α) ^(t)+Be^(−k) ^(β) ^(t)) asdescribed previously [Kronman, C., et al., 2000, ibid]. This modelenables determination of the parameters A and B which represent thefractions of the material removed from the circulation in the first-fastand second-slow elimination phases respectively, and T_(1/2)α andT_(1/2)β which represent the circulatory half-life values of the enzymein the fast and slow phases. The pharmacokinetic parameters MRT (meanresidence time, which reflects the average length of time theadministered molecules are retained in the organism) and CL (clearance,which represents the proportionality factor relating the rate ofsubstance elimination to its plasma concentration (CL=dose/area underthe concentration-time curve), were independently obtained by analyzingthe clearance data according to a noncompartmental pharmacokinetic modelusing the WinNonlin computer program. The comparative results are setforth in Table 3 below.

TABLE 3 Pharmacokinetic parameters ΔC-AChE A (% of T_(1/2)□ B (% ofT_(1/2)β Clearance MRT Preparations total) (min) total) (min) (ml/hr/kg)(min) Non-modified 74 ± 8  3.6 ± 0.6 26 ± 2 44 ± 3 170.4 42 ± 3 AChE- 56± 9 29 ± 6 43 ± 3 390 ± 50 14.2 510 ± 70 PEG-5000 low ratio AChE- 46 ± 328 ± 5 64 ± 3 540 ± 70 13.2 740 ± 80 PEG-5000 high ratio AChE- 35 ± 3 32± 5 65 ± 5  750 ± 130 12  950 ± 120 PEG- 20000 low ratio AChE- 23 ± 4 35 ± 15 76 ± 3 1550 ± 120 4.3 2100 ± 200 PEG- 20000 high ratio

The pharmacokinetic characteristics of the different PEG-AChEpreparations are also set forth in FIG. 2 which shows pharmacokineticprofiles of non-modified and PEG-modified AChE.

As it is seen from the results reported above, in all cases circulatoryresidence is significantly improved by PEG conjugation. The mostprominent effect was observed following modification of ΔC-HuAChE witheither PEG-5000 or PEG-20000 at the higher PEG to AChE ratio. In thelatter case, the MRT was 50 times longer than that of the non-modifiedenzyme. Such a high MRT value in ICR mice, exceeds by far most of thepreviously reported values for different types of AChE or BChE moleculesfrom either recombinant, native or serum derived origin [Kronman, C., etal. (1995) Biochem. J. 311, 959-967; Saxena, A., et al. (1997) Biochem.36, 7481-7489].

The MRT values of the various PEG-modified AChE preparations (differingin the number and size of the appended PEG molecules), as presented inTable 3, is linearly dependent on the overall apparent molecular weightof the PEGylated-HuAChE preparation (the “average” apparent molecularweight for each of the preparations was computed by determination of therelative abundance of PEGylated products in a given preparation bysubjecting its SDS-PAGE Western blots profile to densitometricanalysis). The finding of the linear relationship in FIG. 3 suggeststhat the factor determining the circulatory time of AChE, is not thenumber of the modified sites per se, but the actual increase inmolecular size as a consequence of the PEGylation. Thus, it appears thatthe attachment of a single very large PEG unit to HuAChE may be asefficient as PEGylation of all potential lysines by smaller PEGsubunits.

Example 5

A circulatory AChE form purified from fetal bovine serum (FBS-AChE) wasemployed for conjugation. This enzyme form represents, therefore, anative version of AChE, which, by virtue of its serum origin, exhibits acirculation residence ability superior to recombinant forms of theenzyme both human or bovine (see below). FBS-AChE differs from therecombinant human AChE of Example 4, in several respects: (i) the aminoacid composition of bovine AChE differs from its human counterpart by 34amino acids, including 2 lysines (out of 10) that are present in HuAChEbut are missing in BoAChE [Mendelson, I., et al. (1998) Biochem. J. 334,251-259], (ii) the bovine version of the enzyme contains four sites ofN-glycosylation rather than three exhibited by the human form[Mendelson, I., et al., 1998, ibid], (iii) the native FBS-AChE displaysfully sialylated glycan termini (Kronman, C., et al., 2000, ibid), (iv)as is set forth in FIG. 5, FBS-AChE is tetrameric in nature whereas theC-terminal truncated version of AChE (Example 4) is monomeric(analytical sucrose density gradient centrifugation was performed asdescribed [Kronman, et al., 1995, ibid]) and (v) FBS-AChE displays a MRTof 1340 min (compare to the truncated rHuAChE from Example 4 whichexhibits, in its non-PEGylated state, a MRT of 42 min). FBS-AChE waspurified from serum as described in Example 1. The purified enzyme wasconjugated to SPA-PEG as described in Example 2. Clearance experimentsin mice and analysis of pharmacokinetic profiles were carried out asdescribed in Example 4. The comparative pharmacokinetic profiles ofnon-modified and PEG modified FBS-AChE are set forth in FIG. 4. From theforegoing it is clearly seen that modification of FBS-AChE by PEGsignificantly improved its pharmacokinetic behavior. Moreover, togetherwith Example 4, it is clear that the improvement of circulatorylongevity by PEG modification is not dependent on the origin of theenzyme (e.g. recombinant or native; human or bovine). The findings thatmodification of both forms by PEG resulted in a long lived enzyme(Example 4 and this example), indicates that conjugation of PEGincreases circulatory retention regardless of amino-acid sequencedivergence, number of appended glycans, level of N-glycan terminalsialylation and oligomeric nature of the enzyme.

Example 6

The pharmacokinetic improvement of AChE promoted by PEG-conjugation wasstudied in conjunction with the terminal sialylation level of theN-glycans appended to the enzymes. The way sialylation andPEG-modification may interplay in determining serum residence time ofAChE is of special importance due to the crucial role of efficientsialylation in preserving circulatory longevity of AChE (Kronman, C., etal., 1995, ibid; Chitlaru, T., et al., 1998, ibid; Kronman, C., et al.,2000, ibid), probably involving the hepatic asialoglycoprotein receptorwhich efficiently mediates rapid clearance of undersialylated (bearingexposed gal residues) glycoproteins [Ashwell, G. and Harford, J. (1982),Ann. Rev. Biochem. 51:531-554]. Notably, in many recombinant systemshigh level of heterologous glycoprotein production is associated withlow level of sialic acid capping. Indeed, it has been documented that adirect correlation exists between the level of AChE production and theextent of N-glycan terminal sialylation, resulting in severeundersialylation and hence, poor pharmacokinetic performance of rHuAChEgenerated by high producer clones [Chitlaru, T., et al., 1998, ibid]. Inthis example therefore, non-sialylated or fully-sialylated AChE formswere used for PEG conjugation. Recombinant AChE was purified from tissueculture medium as described in Example 1. Generation of desialylatedrHuAChE was achieved by subjecting the purified enzyme to treatment withsialidase (neuroaminidase) as described before [Chitlaru, T., et al.,1998, ibid]. Generation of fully sialylated rHuAChE was achieved byexpressing the HuAChE gene in the genetically modified 293ST-2D6 cellswhich stably express a recombinant rat Golgi-version of 2,6sialyltransferase [Chitlaru et al., 1998, ibid, Kronman et al., 2000,ibid]. The fully sialylated AChE was purified from tissue culture mediumas described in Example 1. The sialidase-treated or the fully sialylatedenzymes were conjugated to PEG as described in Example 2. Glycanstructures of these AChEs were determined by MALDI-TOF analysis asdescribed in the art [Kronman, C., et al., 2000, ibid]. Analysis ofglycan structures and level of sialylation is set forth in FIG. 6.Clearance experiments in mice and analysis of pharmacokinetic profileswere carried out as described in Example 4. The comparativepharmacokinetic profiles are set forth in FIG. 7. From the foregoing itis clearly seen that modification of the sialidase-treated or fullysialylated AChE by PEG significantly improved their pharmacokineticbehavior. Moreover, it is clear that the improvement of circulatorylongevity by PEG modification is not dependent on the nature of theappended glycans of the enzyme, and that PEG conjugation can rescue anextremely circulatory short lived form of the enzyme, as well ascompensate for low level of sialic acid occupancy which ispharmacokinetically deleterious.

Example 7

Recombinant human AChE which carries one, instead of three appendedglycans, or which is completely non-glycosylated, was used forconjugation. The ability of PEG to promote pharmacokinetic improvementof AChE with a reduced number of appended N-glycans is of specialinterest in view of the fact that recombinant proteins generated inbacterial systems do not contain glycans. To examine the effect ofPEG-conjugation on AChE carrying a reduced number of N-glycanappendages, a mutated recombinant human AChE (N350Q/N464Q) which resultsin the generation of AChEs harboring one glycan per molecule was usedfor conjugation. The recombinant AChE was purified from tissue culturemedium as described in Example 1. To generate AChE which is completelydevoid of N-glycans, recombinant AChE was generated and purified fromtissue culture medium as described in Example 1. The purified enzyme wastreated with N-glycanase to remove all N-linked glycan structures.Recombinant AChE (250-500 μg of either the wild-type or the C-terminaltruncated enzyme) was subjected to treatment with 250 mU of N-glycanase(Glyko Inc. or Boehringer Manheim GmbH) at room temperature for 48hours. N-glycanase was removed by subjecting the treated enzyme to asecond round of purification as described in Example 1. The completeremoval of the glycans from AChE was monitored by SDS-PAGE analysis andis set forth in the inset of FIG. 8. The monoglycosylated AChE and thenon-glycosylated enzyme were conjugated to PEG as described in Example2. Clearance experiments in mice and analysis of pharmacokineticprofiles were carried out as described in Example 4. The comparativepharmacokinetic profiles of non-modified and PEG-modifiednon-glycosylated AChE are set forth in FIG. 8. The comparativepharmacokinetic profiles of non-modified and PEG-modifiedmono-glycosylated AChE are set forth in FIG. 10. From the foregoing itis clearly seen that modification of the monoglycosylated ornon-glycosylated AChE by PEG significantly improved theirpharmacokinetic behavior. These results show that the improvement ofcirculatory longevity by PEG modification is not dependent on thepresence or the quantity of appended glycans of the enzyme.

Example 8

A mutated recombinant human AChE (N350Q/N464Q) was used for conjugation.Notably, the mutations N350Q and N464Q of this form of enzyme result inthe generation of AChEs harboring one glycan per molecule. Therecombinant AChE was purified from tissue culture medium as described inExample 1. The glycan structures of this mutant were determined byMALDI-TOF analysis as known in the art [Kronman, C., et al., 2000,ibid]. The glycan analysis is set forth in FIG. 9. The glycan analysisrevealed that approximately 50% of the glycans appended to this mutantform of AChE are of the high-mannose type. In addition to the highmannose type of N-glycans associated with this form of enzyme, theMALDI-TOF analysis revealed the presence of a substantial fraction ofmolecules harboring immature glycans (terminating in GalNAc, see FIG.10). The purified enzyme was conjugated to PEG as described in Example2. Clearance experiments in mice and analysis of pharmacokineticprofiles were carried out as described in Example 4. The comparativepharmacokinetic profiles are set forth in FIG. 10. From the foregoing itis clearly seen that modification of this mutant AChE by PEGsignificantly improved its pharmacokinetic behavior.

Example 9

Equine BChE (accession number AAF61480) was subjected to lysine-directedmodification by PEG, followed by assessment of its pharmacokineticbehavior. Equine BChE was purified from horse serum by affinity toprocainamide as described in Example 1. BChE was partially desialylatedby enzymatic treatment with neuraminidase as described in Example 6,subjected to PEG conjugation as described in Example 2 and administeredto mice for pharmacokinetic characterization as described in Example 4.Unlike the non-treated serum-derived BChE which resides in thecirculation for extended periods of time, the partially desialylatedenzyme displays a dramatically shortened circulatory residence time(MRT=160 min, see FIG. 11). However, when this sub-optimally sialylatedenzyme was subjected to PEG attachment, the circulatory residence timewas significantly increased, to levels commensurate with those exhibitedby the native, long-lived enzyme.

Although belonging to the same family of cholinesterases as AChE, BChEdiffers from human AChE by more than 330 amino acids. Most notably, theequine version of BChE contains 33 lysine residues and 9 glycans perenzyme subunit as opposed to 7 lysine residues and 3 glycans present inthe truncated version human AChE. The ability to extend the circulatoryresidence of equine butyrylcholinesterase illustrates the feasibility ofPEG-modification procedure to generate long-lived OP-bioscavengers froma wide variety of cholinesterases differing in their source, primarysequence, lysine and glycan contents and enzymatic specificities.

1. A soluble, circulatory long-lived organophosphate scavenger with amean residence-time (MRT) in the body of at least 500 minutes, whichscavenger is a recombinant cholinesterase (ChE) protein conjugated topolyethylene glycol (PEG); said ChE being selected fromacetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and saidPEG having molecular weight of from about 5000 to about 20,000 dalton;wherein the number of PEG molecules conjugated to one ChE moleculeranges from 1 to 11 for said AChE, and from 1 to 40 for said BChE. 2.The scavenger of claim 1 wherein the recombinant AChE is mutated at oneor more amino-acid residues.
 3. The scavenger of claim 1 lwherein therecombinant BChE is mutated at one or more amino-acid residues.
 4. Thescavenger of claim 1 wherein the polyethylene glycol ismono-methoxy-PEG.
 5. The scavenger of claim 4 wherein the PEG ischemically-activated-PEG.
 6. The scavenger of claim 5 wherein thechemically-activated-PEG is succinimidyl derivative of PEG propionicacid (SPA-PEG).
 7. The scavenger of claim 1 wherein the PEG isconjugated through primary amines, carboxyl sites, or thiol groups, orcarbohydrates.
 8. A pharmaceutical preparation comprising the scavengerof claim 1.